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Developmental and Persistent Toxicities of Maternally Deposited Selenomethionine in Zebrafish (Danio rerio) Jith Kallarakavumkal Thomas, and David M. Janz Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02451 • Publication Date (Web): 21 Jul 2015 Downloaded from http://pubs.acs.org on July 25, 2015
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Environmental Science & Technology
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Developmental and Persistent Toxicities of Maternally Deposited
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Selenomethionine in Zebrafish (Danio rerio)
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Jith K. Thomasa and David M. Janz b,c *
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a
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S7N 5B3
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b
Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B3
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c
Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon,
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Saskatchewan, Canada S7N 5B4
Toxicology Graduate Program, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
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*Corresponding author: David M Janz
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Tel. 1 -306-966-7434
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Fax. 1-306-966-7376
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Email address:
[email protected] 1 ACS Paragon Plus Environment
Environmental Science & Technology
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ABSTRACT
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The objectives of this study were to (1) establish egg selenium (Se) toxicity thresholds for
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mortality and deformities in early life stages of zebrafish (Danio rerio) after exposure to excess
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selenomethionine (SeMet; the dominant chemical species of Se in diets) via in ovo maternal
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transfer, and (2) investigate persistent effects of developmental exposure to excess SeMet on
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swim performance and metabolic capacities in F1 generation adult zebrafish. Adult zebrafish
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were fed either control food (1.3 µg Se/g, dry mass or d.m.) or food spiked with increasing
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measured concentrations of Se (3.4, 9.8 or 27.5 µg Se/g, d.m.) in the form of SeMet for 90 d. In
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ovo exposure to SeMet increased mortality and deformities in larval zebrafish in a concentration-
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dependent fashion, with threshold egg Se concentrations (EC10s) of 7.5 and 7.0 µg Se/g d.m.,
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respectively. Impaired swim performance and greater respiration and metabolic rates were
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observed in F1 generation zebrafish exposed in ovo to 6.8 and 12.7 µg Se/g d.m and raised to
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adulthood in clean water. A species sensitivity distribution (SSD) based on egg Se
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developmental toxicity thresholds suggests that zebrafish are the most sensitive fish species
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studied to date.
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INTRODUCTION Although trace concentrations of selenium (Se) are required to maintain physiological
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homeostasis in all animals, a marginal increase in dietary intake of Se can lead to
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bioaccumulation and subsequent toxicity.1 It has been widely recognized that oviparous species,
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especially fishes, are highly susceptible to toxic effects of Se.1,2 Comparison of the range
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between essentiality and toxicity of ingested trace elements in freshwater fishes clearly 2 ACS Paragon Plus Environment
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demonstrates that Se is the most toxic essential trace element yet studied, with a narrow margin
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of safety.1,3 Selenium is classified as a contaminant of potential concern in many countries due
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to poisonings reported in fishes and other wildlife.4,5 Anthropogenic activities such as mining,
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coal-fired power plants, oil refining activities, and agriculture on seleniferous soils can
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significantly augment mobilization of Se into aquatic systems.4,5 Selenium enters aquatic
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ecosystems mainly as selenate and selenite, where primary producers and certain bacteria
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facilitate biotransformation of these inorganic forms into more bioavailable organoselenium
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forms.6,8 Selenomethionine (SeMet) has been identified as the major form of organic Se present
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in invertebrates6,8 and fishes7,8 collected from Se contaminated aquatic ecosystems, and has
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greater bioaccumulative and trophic transfer properties than other forms of Se.6,8 Both
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laboratory9,10,11 and field studies 12,13 demonstrated that exposure of adult female fishes to excess
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dietary Se (mainly SeMet) can cause greater accumulation of Se in their eggs, and such an
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exposure route (maternal transfer) can augment Se-induced mortality and deformities in early life
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stages of F1 generation fishes.
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Since maternal transfer is the major pathway of Se exposure in early life stages of fishes, in
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2004 the United States Environmental Protection Agency (USEPA) proposed a chronic whole-
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body Se criterion of 7.91 µg/g dry mass (d.m.) for protection of fish populations.14 However,
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subsequent studies12,13 have demonstrated that not a whole body, but an egg or ovary tissue based
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Se criterion may provide better protection against Se-induced developmental toxicities in fishes.
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Lemly15 recommended an egg/ovary Se threshold of 10 µg/g d.m. for protection of freshwater
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fishes, whereas DeForest et al.16 suggested that coldwater species might be less susceptible to
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developmental Se toxicities when compared to warmwater fishes, and recommended an
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egg/ovary Se threshold of 20 µg/g d.m. for protection of coldwater fishes. A more recent 3 ACS Paragon Plus Environment
Environmental Science & Technology
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USEPA Se draft criterion proposed an egg/ovary Se concentration of 15.2 µg/g d.m. for
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protection of freshwater fish populations.17 However, there still exists debate among aquatic
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toxicologists whether the current proposed egg/ovary Se guideline provides sufficient protection
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against Se-induced developmental toxicities in fishes. Previous studies11,18 suggested that
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developmental toxicities in zebrafish (Danio rerio) could occur at egg Se concentrations closer
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to or lower than the current proposed egg/ovary Se draft criterion proposed by the USEPA.
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Although zebrafish is not a native North American species, it is a member of a large,
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economically and ecologically important family of fishes (Cyprinidae). Approximately 270
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cyprinid species have been identified in North America, many of which are listed as threatened,19
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however there are few data available for developmental Se toxicity in cyprinids. Therefore, the
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present study used a cyprinid fish, zebrafish, to investigate immediate and persistent effects of
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developmental exposure to excess SeMet. The objectives of this study were to establish egg Se
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toxicity thresholds for mortality and deformities in early life stages of zebrafish after exposure to
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excess SeMet via in ovo maternal transfer, and to investigate persistent effects of developmental
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exposure to excess SeMet on swim performance (Ucrit), oxygen consumption (MO2) and
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metabolic capacities in F1 generation zebrafish raised to adulthood in clean water. In addition,
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the egg Se toxicity threshold (effect concentration [EC])10 from the present study and other
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studies were used to generate a species sensitivity distribution (SSD) to examine the relative
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sensitivity of zebrafish to Se-induced early life stage toxicity.
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MATERIALS AND METHODS
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Test compound: Seleno-L-methionine was purchased from Sigma-Aldrich (Oakville, ON, Canada). Purity of the compound was greater than 98%.
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Environmental Science & Technology
Diet preparation and adult exposure: All methods used in the present study were approved
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by the University of Saskatchewan’s Animal Research Ethics Board (protocol # 20030076), and
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adhered to the Canadian Council on Animal Care guidelines for humane animal use. Wild type
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(WT) zebrafish were purchased from AQUAlity Tropical Fish Wholesale (Mississauga, ON,
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Canada) and housed in an environmental chamber with controlled temperature (28.0 ± 1.0 °C)
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and photoperiod (14 h light and 10 h dark). Adult fish were acclimated to these laboratory
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conditions for 3 weeks. During the acclimation period fish were fed Nutrafin® basic flake food
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(Hagen Inc., Montreal, QC, Canada). Both diet preparation and dietary SeMet exposure to adult
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fish were described previously.20 Briefly, adult zebrafish were fed twice daily (5% body mass/d
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ration) with either control (1.3 µg Se/g d.m.) or SeMet-spiked diets (3.4, 9.8 and 27.5 µg Se/g
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d.m.) for 90 d. After 90 d of feeding exposure, sub-groups of adult fish from each dietary
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treatment group were bred once to collect eggs for the experiment. Resulting embryos were used
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to investigate adverse effects of excess SeMet exposure via in ovo maternal transfer in F1
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generation fish. Concentrations of total Se in eggs, percent egg viability and hatchability, and
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percent mortality and total deformities in F1 generation larval fish were determined.11
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In addition, a sub-group of larval zebrafish from each treatment group were reared in clean
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water (temperature 28.0 ± 1.0 °C) and fed the control diet until 180 days post fertilization (dpf),
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the mature adult fish stage, to investigate persistent effects of developmental SeMet exposure on
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Ucrit, MO2 and metabolic capacities in F1 generation adult zebrafish.
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Deformity analysis: Total deformity analyses were carried out on 6 dpf larval zebrafish. In
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zebrafish, yolk resorption is completed by 6 dpf 21 and hence we chose 6 dpf for deformity
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analysis since Se assimilation by the developing larvae should then have occurred. The detailed
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larval zebrafish preservation and analysis protocol for deformity assessments was explained 5 ACS Paragon Plus Environment
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previously.11 Severely deformed larval zebrafish were euthanized and preserved on 4 and 5 dpf
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and included in deformity analysis with 6 dpf larval fish. All preserved larval zebrafish were
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examined for malformations in a blind fashion using an Olympus model SZ-CTV dissecting
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microscope (Olympus, Melville, NY, USA). Each larval fish was examined for skeletal,
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craniofacial and fin deformities, and edema, and the presence or absence of developmental
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abnormalities was recorded for each fish. Total percent deformities were calculated by dividing
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number of malformed larval fish by the total number of larval fish, and multiplying by 100.
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Quantification of selenium: Concentrations of total Se in pooled egg samples were
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measured by use of inductively coupled plasma-mass spectrometry (ICP-MS) at the Toxicology
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Centre (University of Saskatchewan, Saskatoon, SK, Canada). For the quantification of Se we
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collected 3 replicates of 100 pooled egg samples from adult female zebrafish fed the control diet
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and 3 replicates of 60 pooled egg samples from adult female fish fed the SeMet spiked diets.
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The detailed Se analysis procedure was described previously.20,22 A limit of quantification
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(LOQ) of 0.3 µg Se/g was determined using method blanks. Concentrations of Se in eggs were
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measured on wet mass basis and converted to dry mass based on a moisture content of 93.7 %.
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The zebrafish egg moisture content was determined in a previously published study.11
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Oxygen consumption and swim performance: Oxygen consumption and swim
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performance were conducted in a modified Blazka-type, variable speed, miniature swim tunnel
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respirometer with a DAQ-M control device and AutoRespTM 1 software (Loligo Systems, Tjele,
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DK). The system consists of a 170 mL swim tunnel submerged in a 20 L buffer tank supplied
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with 28.5 ± 0.1 °C aerated water from a 20 L heated water bath circulator (VWR International,
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Mississauga, ON, Canada). Concentrations of oxygen (O2) were measured using a fiber optic
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oxygen dipping probe which was connected to a Fibox 3 minisensor oxygen meter (Precision 6 ACS Paragon Plus Environment
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Sensing GmbH, Regensburg, DE). AutoRespTM 1 software was used to calculate MO2. The
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detailed MO2 measuring principle is explained elsewhere.23 We adopted a method called critical
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swimming speed (Ucrit) to study aerobic swimming capacity of fish.24 The detailed procedures
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for both oxygen consumption and swim performance were discussed elsewhere.20 Swim
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performance values (cm/s) were corrected for standard body length of individual fish, and thus
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Ucrit data were represented as body lengths per second (BL/s).
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Determination of standard metabolic rate (SMR), active metabolic rate (AMR), factorial
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aerobic scope (F-AS) and cost of transport (COT): Determination of standard metabolic rate
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(SMR), active metabolic rate (AMR), factorial aerobic scope (F-AS) and cost of transport (COT)
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were explained previously.20
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Statistical analysis: All data were tested for normality by use of the Shapiro–Wilk test and
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homogeneity of variance by use of Levene’s test (SigmaStat 3.1, SPSS Inc., Chicago, IL, USA).
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Data that did not meet the assumptions for parametric statistical procedures were log 10
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transformed. Non-transformed data are shown in all Figures. Significant differences in total Se
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concentrations in eggs, egg viability and hatchability, mortality and total deformities of larval
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fish, Ucrit, MO2 and COT at individual water velocities, SMR, AMR, F-AS and morphometrics
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(total length, body mass and condition factor) of adult zebrafish among different treatment
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groups were tested by use of one-way ANOVA followed by the Holm–Sidak post hoc test. Data
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were expressed as mean ± S.E.M. Differences were considered statistically significant at p
